人源神经干细胞来源外泌体的提取鉴定及内吞作用-东南大学学报医学版

人源神经干细胞来源外泌体的提取鉴定及内吞作用

单位：1. 东南大学附属中大医院, 神经外科, 江苏南京 210009;
2. 东南大学医学院, 江苏南京 210009;
3. 东南大学附属中大医院, 妇产科, 江苏南京 210009

分类号：R741.05

出版年·卷·期（页码）：2017·36·第六期（984-989）

摘要：

目的：细胞外囊泡外泌体内含有蛋白、脂质和遗传物质，在细胞间的交流中发挥着非常重要的作用。本研究首次探讨人源神经干细胞来源外泌体的的生物学特性和潜在功能。方法：经东南大学附属中大医院临床研究伦理委员会的审查和批准，我们利用成功分离出的人胎儿海马来源的原代神经干细胞进行体外培养、外泌体获取以及功能评价。通过免疫荧光、western blotting和琼脂糖凝胶电泳对人源神经干细胞进行鉴定；利用超高速离心和总外泌体提取试剂盒提取培养细胞上清中的外泌体，并通过透射电镜、纳米颗粒追踪分析和western blotting进行鉴定；最后用PKH67标记外泌体后评价其潜在功能。结果：人胎儿的海马中原代神经干细胞成功分化为神经元、星形胶质和少突胶质细胞；随后在干细胞的培养上清中成功分离出了干细胞分泌的外泌体，符合外泌体的基本特征结构；最后发现外泌体可以被干细胞摄取和内吞，明显聚集于胞质内，且部分入核。结论：本研究初步探索了人源神经干细胞来源外泌体的基本特征和内吞作用，为其后续在神经系统领域的深入研究奠定了基础。

Objective: Extracellular vesicles (EVs) such as exosomes containing protein, lipid and genetic material, play an important role in the cell-cell communication. This study was the first time to explore the biological characteristics and potential functions of human neural stem cells (hNSCs) derived exosomes. Methods: Approved by the Institutional Review Board of Zhongda Hospital, Southeast University, we isolated primary NSCs from human fetus hippocampus, and extracted hNSCs-derived exosomes for investigation. hNSCs were identified with immunofluorescence, western blotting and agarose gel electrophoresis. Exosomes were extracted from the culture supernatant of hNSCs by ultracentrifugation or Invitrogen's total exosome isolation system, and verified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. Finally the functions of exosomes were investigated under PKH67 labeling. Results: Firstly, primary human NSCs were successfully isolated, which then differentiated into neuron, astrocyte and oligodendrocyte. Also hNSCs-derived exosomes were successfully extracted from the cell culture supernatants, and these exosomes had all of the characteristics of exosomes. Finally, it was found that the exocrine could be absorbed and ingested by the stem cells, and concentrated in the cytoplasm and partly into the nucleus. Conclusion: This study preliminarily explored the basic characteristics and endocytosis of human NSCs derived exosomes, which will lay the foundation for theirs future study in the neurological field.

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